1. Field of the Invention
This invention relates to a stable hemoglobin-based oxygen-carrying composition and methods to store or process same, said hemoglobin composition including hemoglobin, modified hemoglobin and encapsulated hemoglobin.
2. Description of Related Art
Although human whole blood and packed red cell preparations have long been used to restore body oxygen-carrying function, hemoglobin-based compositions are now seen to offer several advantages over these currently standard hemotherapies. Patient safety would be enhanced by eliminating untoward reactions to blood group antigens, improving the maintenance of systemic acid-base and electrolyte balance, and avoiding exposure to blood contaminated with diseases such as AIDS and hepatitis. The high costs of testing blood for blood group antigens and disease factors would also be avoided. Further, hemoglobin-based oxygen-carrying substitutes are expected to have prolonged stability during storage so a more stable supply should be realized.
While a number of parameters may be measured to characterize hemoglobin solution stability, such as oxygen carrying capacity and ionic composition, the most important of these is the oxidation state of hemoglobin iron. The iron atom in each of the heme prosthetic groups of the hemoglobin molecule is the site of oxygen binding and release. In order to maintain this reversible oxygen binding capability, the heme iron must be in the physiological Fe.sup.2+ state. When a solution of hemoglobin is stored for a long period of time, the iron tends to oxidize to the Fe.sup.3+ state, giving the methemoglobin form which does not reversibly bind oxygen and is therefore physiologically ineffective. (H. F. Bunn et al. Hemoglobin: Molecular Genetic and Clinical Aspects 640 (1986)).
One current strategy to prevent the above described hemoglobin autoxidation reaction is the addition of anti-oxidants (Stratton, L. P., Hemoglobin 12(4):353-368(1988)) such as NADH (Kajita, A. et al., Biochem. Biophys. Res. Comm. 39:1199(1970)). However, in hemoglobin solutions some anti-oxidants can become pro-oxidants (ibid. Stratton et al.). Further, the addition of a component to an intravenous solution product complicates manufacture and raises issues of the toxicity of the new component which can require costly testing during development.
The active reduction of methemoglobin in solution can be accomplished by electrochemical means (P. Scheller, Mechanism of Cathodic Reduction of Hemoproteins, 60 Studia Biophysica 137 (Berlin) (1976)), the use of reductive enzyme systems (Hayashi, A. et al. Biochem. Biophys. Acta 310:309(1973)) or the addition of chemical reducing agents such as ascorbate (Tomoda, A. et al., J. Biol. Chem. 253 (20):7415-7419 (1978)). These systems all require chemical additives and suffer from the same limitations mentioned above for anti-oxidants.
A viable hemoglobin based oxygen-carrying composition should contain less than 50% and preferably less than 15% methemoglobin over the shelf life. The rate of hemoglobin auto-oxidation is, of course, dependent on temperature. Thus, at room temperature (25.degree. C.) heme iron oxidation can exceed 30% per day (See Devenuto, F., infra at 946). Consequently, hemoglobin based oxygen carrying compositions are typically stored in the frozen state to obtain the targeted shelf-life.
The extent of hemoglobin protein purity is an important consideration in any discussion of hemoglobin stability in solution. Hemoglobin heme iron is continuously oxidized in vivo and the red cell contains enzyme systems that directly reduce methemoglobin to restore hemoglobin function. (Hultquist, D. E. et al., The Methemoglobin Reduction System of Erythrocytes, in The International Conference on Red Cell Metabolism and Function, 297-301; Univ. of Michigan (1974)). Paul, H. B. et al, Preparation of Hemoglobin Solutions for Intraveneous Infusions, 455, 463 (1947). Other red cell enzymes eliminate activated oxygen products, such as superoxide and hydrogen peroxide, that can also oxidize heme iron. (Watkins J. A. et al., Biochem. Biophys. Res. Comm. 132(2):742-748 (1985)). It is not surprising therefore that less pure preparations of hemoglobin exhibit increased oxidation stability, particularly when the native methemoglobin reductase substrates, such as NADH, are added to the preparation (Stratton, L. P. et al., Hemoglobin 12(4):353-368 (1988)). Despite this oxidative protection in impure preparations, contaminating proteins can cause immunogenic reactions in patients or may possess innate toxic properties when free in the circulation. Thus, a system that could provide oxidation stability and/or reduce methemoglobin in purified hemoglobin preparations, over a broad temperature range, would possess a substantial advantage.